Glucocorticoids were found to bind to two components in the AtT-20/D-1 pituitary tumor cell. One component was characterized by slow dissociation of the bound steroid, stringent glucocorticoid specificity and high steroid binding affinity (Kd = 4.64 - 10(-9) M for triamcinolone acetonide). Thus, the characteristics of this component, termed the slowly dissociable component, resembled those of the soluble cytosol receptor. The other component exhbited lower binding affinity (Kd = 1.57 - 10(-7) M for triamcinolone acetonide), less stringent glucocorticoid specificity, and very rapid dissociation of bound, labelled glucocorticoid, Binding to this component, termed the rapidly dissociable component, represented 60% of total steroid binding to intact cells at 4 degrees C. Incubation of intact cells at 25 degrees C caused a progressive increase in steroid binding to the slowly dissociable component with no change in the absolute amount of ste roidal binding to the rapidly dissociable component. The high-binding affinity and preference for glucocorticoids shown by both components favor their function as biologically significant mediators of steroid action in this glucocorticoid responsive cell.